Most cancer patients die from metastatic disease as the result of circulating tumor cells (CTCs) spreading from a primary tumor through the blood to distant organs. Clinical studies have demonstrated the tremendous potential of using CTC counts as prognostic or predictive markers of metastatic development and therapeutic efficacy. Despite significant progress in the development of in vitro CTC assays, they still have low sensitivity due to the small samples of blood taken. To overcome these problems, we developed in vivo photoacoustic (PA) flow cytometry (PAFC) and have demonstrated its feasibility in the first pilot trials in melanoma patients. However, these trials revealed the principal limitations nt only of PAFC but most optical diagnostic and therapeutic methods in vivo. These include poor delivery of noninvasive optical radiation to deep tissue, laser safety concerns, and little progres in early cancer diagnosis, prevention metastasis, and overcoming of tumor resistance. Simultaneously, these trials helped identify innovative ways to further significantly improve optical technology in vivo, particularly PAFC, including the safe delivery of a higher level of lasr energy to deeper tissue and more efficient detection of low pigmented CTCs in complex biological backgrounds. The goal of this proposal is to develop innovative approaches to significantly enhance the sensitivity of optical methods in vivo, with a focus on the PAFC platform, and to demonstrate its capabilities in a large cohort of melanoma patients for real-time control of therapeutic efficacy through detection and identification of CTCs, and potentially CTPs. In particular, we propose the concept of fractionated delivery of diagnostic laser energy, nonlinear nanobubble-associated signal amplification in humans, picosecond spectroscopy with switchable wavelength laser, controlling of CTC sizes, and a focused spherical ultrasound transducer array, together with optical skin clearing to reduce light scattering in human skin. To achieve this goal, we will pursue the following Specific Aims: (1) Test the concept of nonlinear, fractionated delivery of laser energy. (2) Optimize the parameters of the nonlinear, fractionated PA detection and PT eradication of CTCs in vitro. (3) Explore the clinical capabilities of in vivo nonlinear, fractionated PA diagnosis and PT therapy of CTCs in melanoma patients during and after conventional treatment. The new clinical prototype of label-free PA-PT theranostic platform will be tested in vivo in humans in three stages: (1) in vivo study of healthy individuals, 2) in vvo PA-PT theranostics in advanced-stage melanoma patients, and 3) controlling CTCs at surgery and during drug administration in melanoma patients. In the course of this study, we will obtain statistically significant data that will demonstrate this innovative technique's unprecedented sensitivity threshold of 1 CTC/500 mL; we will also attempt to achieve a threshold of 1 CTC/1-3 L.